Hymenoptera, Pteromalidae) from Black Locust

bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Title:

3 A new species of Mesopolobus Westwood (Hymenoptera, Pteromalidae) from black locust

4 crops

6 Authors:

8 László, Zoltán1, Lakatos, K. Tímea2, Dénes, Avar-Lehel1, 3

10 Author affiliations:

12 1Hungarian Department of Biology and Ecology, Babeş-Bolyai University, Str. Clinicilor nr. 5–7,

13 400006 Cluj-Napoca, Romania. e-mail: [email protected]

15 2Department of Ecology, University of Debrecen, Debrecen, Egyetem square 1, H-4032, Hungary.

16 e-mail: [email protected]

18 3Interdisciplinary Research Institute on Bio–Nano–Sciences of Babe–Bolyai University, Treboniu

19 Laurian 42, 400271, Cluj-Napoca Romania. e-mail: [email protected]

21 Corresponding author:

23 László, Zoltán, Department of Biology and Ecology, Babeş-Bolyai University, Str. Clinicilor nr. 5–

24 7, 400006 Cluj-Napoca, Romania. e-mail: [email protected] 1

26 Abstract

28 A new species of the genus Mesopolobus Westwood, Mesopolobus robiniae sp. n., is described and

29 illustrated from east-central Europe (Romania and Hungary). The species was reared from black

30 locust (Robinia pseudoacacia) seedpod samples, where it most likely parasitizes the black locust’s

31 seed predator Bruchophagus robiniae Zerova, 1970. Here we present the new species and report on

32 its ecological relationships within the European seed predator community of black locust. We also

33 give details regarding type material and type locality, a detailed description with images, a

34 differential diagnosis of the new species, and a modification to the identification key published by

35 Graham (1969), that distinguishes this new species from closely related species. In addition, we

36 provide information on the distribution, biology and results of barcoding analysis. We also provide

37 the DNA sequence data to complement the morphological taxonomy.

39 Keywords: Robinia pseudoacacia, Bruchophagus robiniae, Mesopolobus, parasitoid, new species

41 Introduction

43 In the last century the black locust (Robinia pseudoacacia L.) became a characteristic component

44 feature of the Central and Eastern European landscape (Vítková et al., 2017). Its positive economic,

45 but negative environmental impacts led to conflicts between nature conservationists, forestry

46 workers, urban planning experts, beekeepers and the public (Benesperi et al., 2012; Dickie et al.,

47 2014; Sádlo et al., 2017). As current legislation will determine the future distribution of black

48 locust, we need detailed knowledge, not only from the viewpoint of the forestry and economy, but 2

49 also from the viewpoint of the species potential associates, like herbivorous insects and their

50 community (Kleinbauer et al., 2010).

52 The invasive history of black locust follows the characteristic pathway of introduced crops with an

53 initial phase when presumably several independent introductions occurred from North America,

54 which ceased for a long period, then were followed by frequent plantings and a rapid invasion in the

55 wild, resulting in its widespread distribution of today (DAISIE, 2009). The invasion of black locust

56 in Central and Eastern Europe was facilitated by extensive plantings, due to the wood’s long-term

57 quality, resistance to insects and fungi, rapid growth, easy propagation, and ability to stabilize soils

58 (Vítková et al., 2017).

60 When replacing native vegetation, the black locust reduces local biodiversity (Hanzelka & Reif,

61 2015). Endangered light-demanding plants and invertebrates are threatened by its appearance

62 through reducing light to plants growing beneath the canopy and above the forest floor, and

63 changing the microclimate and soil quality (Lazzaro et al., 2018). These impacts can have effects

64 throughout the food chain, by depriving birds of their insect prey, which depend on the plants that

65 have been wiped out by the black locust (Hanzelka & Reif, 2015). One of the central problems

66 regarding black locust colonization is its capacity to rapidly increase soil nutrient concentration and

67 to alter soil chemical properties which conditions then facilitate invasion by other non-native

68 nitrophilous plant species (Enescu & Dănescu, 2013).

70 Because of the wide distribution and negative environmental impacts of black locust an important

71 management tool for invasive populations is limiting their propagation (Redei et al., 2001). The

72 crops of black locust are attacked by several pests, among which the seed predators can have a 3

73 major impact (Zerova, 1970; Perju, 1998). Bruchophagus robiniae Zerova, 1970 (Hymenoptera:

74 Chalcidoidea: Eurytomidae) is a pre-dispersal seed predator, monophagous on black locust seeds

75 (Perju, 1998). As with other Bruchophagus species, each B. robiniae individual feeds and develops

76 inside one infested seed, so each seed wasp consumes only one seed and each seed houses only one

77 seed wasp (Lakatos et al., 2018). This seed predator has a component community comprising

78 several species, including parasitoid wasps (Lakatos et al., 2016; 2018).

80 One member of the Bruchophagus robiniae - black locust seed predator community belongs to the

81 genus Mesopolobus Westwood, 1833, a group of parasitoid wasps in Pteromalidae (Hymenoptera:

82 Chalcidoidea) containing more than 120 described species (Noyes, 2020), over 60 of which are

83 present in Europe (http://www.fauna-eu.org/). Species of this genus have a wide host range,

84 although several species are known to be host-specific on galls (Diptera: Cecidomyiidae,

85 Hymenoptera: Cynipidae), bark beetles (Scolytinae), seed predators (Lepidoptera, Coleoptera:

86 Curculionidae, Bruchinae, Hymenoptera: Eurytomidae), etc. (Bouček & Rasplus, 1991; Noyes,

87 2020). Several Eurytomidae species, such as Bruchophagus gibbus (Boheman, 1836) has

88 Mesopolobus sp. parasitoids (Noyes, 2020), and several Mesopolobus species are parasitoids of

89 pests on oilseed rape (Brassica napus L.), on alfalfa (Medicago sativa L.), or on Norway spruce

90 (Picea abies L.) (Noyes, 2020).

92 Mesopolobus is a taxonomically complex genus, considering the high number of species belonging

93 to this genus. European Mesopolobus were revised by Hans von Rosen (von Rosen, 1958; 1959;

94 1960; 1961), who synonymized species from multiple genera (Amblymerus Walker, 1834; Eutelus

95 Walker, 1834; Platyterma Walker, 1834) under Mesopolobus. The last revision of the Mesopolobus

96 genus for the Western Palearctic was written by Graham (1969). Later studies dealing with 4

97 Mesopolobus parasitoids of certain host groups have provided further clarifications of species

98 synonymies, notably parasitoids of gall inducing Cynipidae on Quercus sp. (Askew, 1961; Aldrey,

99 1983; Pujade-Villar, 1993) and of seed weevils associated with Brassicaceae (Baur et al., 2007).

100 Since the latest generic revision (Graham, 1969) several new species have been described from Asia

101 (e.g. Narendran et al., 2011; Xiao et al., 2016) and North-America (e.g. Doganlar, 1979).

102

103 The identification of Mesopolobus species emerged from black locust crops was based on the most

104 detailed identification key up to date provided by Graham (1969). Using Graham’s keys a number of

105 characters (fore wing marginal vein length ratio to stigmal vein length, number of anelli and

106 funicular segments, position of toruli to anterior margin of clypeus and to median ocellus, pilosity of

107 the basal cell of fore wing, position of hypopygium tip along the gaster) led us to key couplet 16

108 (page 643), where based on two character combinations, namely gaster length ratio to head plus

109 thorax length and gaster breadth, which did not match our specimens. This suggested the specimens

110 reared from the black locust pods were not represented in the keys, and were likely undescribed. We

111 thus studied several Mesopolobus species represented in the keys and compared them

112 morphometrically to the Mesopolobus females emerged from black locust seed pods. This approach

113 provides robust insight into Mesopolobus morphology, which may play a major role in resolving the

114 species delimitations in biocontrol studies. Complementing the morphometric study, we also

115 analyzed mtCOI sequences of the emerged Mesopolobus females from black locust pods, and

116 compared them to the available mtCOI sequences from the BOLD System and NCBI databases.

117

118 Our objectives were the following: i) to identify those morphometric characters that give the best

119 discrimination of the females emerged from black locust seedpods from other Mesopolobus species.

120 ii) to calculate the genetic distance values between the mtCOI sequence of the females emerged 5

121 from black locust and the other Mesopolobus species. iii) to describe the species of the females

122 emerged from black locust seedpods.

123

124 Materials and methods

125

126 To gather information about black locust seedpod insect inhabitants we collected seedpod samples

127 in black locust plantations and patches for four years, in the early spring of 2009 in Romania and

128 between 2013-2015 in Romania and Hungary (Table 1). Samples were placed in plastic cups,

129 containing 20-100 seedpods and covered with punched plastic wrap. Samples were kept in a covered

130 balcony with a temperature and humidity close to outdoors at Babe-Bolyai University (Cluj-

131 Napoca, Romania) and at University of Debrecen (Debrecen, Hungary). Emerged individuals were

132 monitored and collected monthly from seedpod samples for a year, and stored in 70% ethanol. The

133 dominant emerging species were the seed predator of black locust seeds, Bruchophagus robiniae,

134 and its parasitoid, the undescribed Mesopolobus species (Lakatos et al., 2018).

135

136 Identification and description of the emerged Mesopolobus species have been made under an

137 Olympus SZ51 binocular microscope, with an 80X magnification and LED lighting. Images were

138 produced by a Canon EOS 600D and a Canon EF 100mm f/2.8 USM Macro Lens. Morphological

139 nomenclature follows Graham (1969). The provided identification key is modified from the keys to

140 genus Mesopolobus of Graham (1969). Type material is deposited in the Museum of Zoology,

141 Babeş-Bolyai University, Cluj-Napoca (MZBBU). Specimen identification codes: holotype–

142 MZBBU HYM000011; 14 paratypes–MZBBU HYM000012-25, measured female specimens:

143 HYM000026-39.

144 6

145 For morphological comparison several specimens were loaned from different museums. The

146 specimens of Mesopolobus amaenus (Walker, 1834), M. apicalis (syn. thomsonii) (Thompson,

147 1878), M. aspilus (syn. elongates) (Walker, 1835), M. diffinis (Walker, 1834), M. dubius (Walker,

148 1834), M. fasciiventris Westwood, 1833, M. semiclavatus (Ratzeburg, 1848) and M. typographi

149 (Ruschka, 1924) were loaned from the Hungarian Museum of Natural History, Budapest, Hungary

150 (HMNH). Specimens of M. verditer (Norton, 1869), M. mediterraneus (Mayr, 1903), M. tibialis

151 (Westwood, 1833) and M. xanthocerus (Thomson, 1878) were loaned from the British Natural

152 History Museum. One female specimen of M. longicollis Graham, 1969 was measured using ImageJ

153 from photographs of the type provided by Oxford University Museum of Natural History.

154

155 We measured 19 morphometric variables, corresponding to those used in the taxonomy of

156 Pteromalidae for calculating typically used ratios (e.g. Graham, 1969) (Table 2), on a total of 55

157 dry-mounted Mesopolobus females belonging to the above-named species (Supplementary Material:

158 Table S1). Measurements were made with an Olympus SZ51 stereo microscope (objective:

159 110AL2X; eyepiece: WHSZ10X) under 60× and 80× magnification using a calibrated eye-piece

160 micrometer (2.5 mm subdivided into 100 units). For all measurements we ensured that the points of

161 reference were equidistant from the objective of the microscope.

162

163 Body ratios of Mesopolobus female specimens were analyzed using the Multivariate Ratio Analysis

164 (MRA) tool (Baur & Leuenberger, 2011). Variation structure of Mesopolobus specimens was

165 analyzed by PCA in shape space to identify the principal components accounting for the variation.

166 For the visualization of each character’s contribution we used PCA ratio spectrum. Body ratios with

167 best discriminant power were determined using the LDA ratio extractor (Baur & Leuenberger,

168 2011). Analyses were made with R statistical software version 3.6.3 (R Core Team, 2020). 7

169

170 Genomic DNA was extracted from three individuals using DNeasy Blood and Tissue kits (Qiagen

171 Inc., Valencia, CA), following the protocol provided by the manufacturer. Mitochondrial

172 cytochrome c oxidase subunit I (COI) sequences were amplified using the standard LCO1490 and

173 HCO2198 primer pair (Folmer et al., 1994) in a 50 µl reaction volume at a 45°C annealing

174 temperature. PCR products were purified with the Wizard SV Gel and PCR Clean–Up System

175 (Promega, USA) and sent for sequencing to Macrogen Inc. (Korea).

176

177 Sequences were downloaded and verified with the Basic Local Alignment Search Tool (BLAST)

178 (Johnson et al., 2008). Further, sequences for all available Mesopolobus species were also

179 downloaded from the NCBI database and the BOLD System (for reference numbers see Figure 2).

180 The sequences were aligned using a Clustal W algorithm (Thompson et al., 1994) in BioEdit (Hall,

181 1999). A phylogenetic tree was inferred in MrBayes (Ronquist et al., 2012), assuming a GTR+G+I

182 model. Interspecific p-distances were calculated in MEGA X (Kumar et al., 2018).

183

184 Results

185 Multivariate Ratio Analysis of variation in body size and shape

186

187 We first performed a series of shape PCAs on all specimens based on 19 morphometric characters.

188 We identified principal components contributing to morphometric variation of all Mesopolobus

189 females without prior species-determination by applying the PCA in isometry free shape space

190 function to all specimens as a single group. Then we applied the PCA in isometry free shape space

191 only to the group of females which were closest to those emerged from black locust seedpods. When

192 we included all females in shape space, PC1 and PC2 accounted for 58% of the variation of the 8

193 entire sampled population. When analyzing only those species pairs which were closest to our target

194 group in shape space, PC1 and PC2 accounted for 61% and 74% of the variation respectively. The

195 first principal components are congruent with the separation of species, although a clear cut between

196 the clusters could not be established (Figure 1). On the first scatterplot (Figure 1a) only five species

197 (M. amaenus, M. verditer, M. sericeus, M. typographi and Mesopolobus sp. n.) showed a clear

198 separation from the rest, but because of the overplotting with Mesopolobus sp. n we also retained M.

199 fasciiventris for further analyses. On the other two scatterplots (Figure 1b and 1c) the selected

200 species show almost clear separations on PC1, while on PC2 are overlapping.

201

202 The PCA ratio spectrum for the species pair M. amaenus and Mesopolobus sp. n. (Figure 2a)

203 identified ltg.l at the extreme high end, and stv.l at the extreme low end of the spectrum. These

204 characters were also found to contribute to species discrimination. The allometry ratio spectrum for

205 the first species pair was dominated almost by the same ratio, stv.l and ltg.l (Figure. 2b), which is

206 also the most important ratio concerning the first shape PC which shows to be the most allometric

207 one. The PCA ratio spectrum for the species pair M. fasciiventris and Mesopolobus sp. n. (Figure

208 2c) identified pcl.l at the extreme high end, while stv.l at the extreme low end of the spectrum.

209 These characters, except for stv.l, were found to contribute to species discrimination. The allometry

210 ratio spectrum for the second species pair was dominated almost by the same ratio, pcl.l and ltg.b

211 (Figure. 2d), that is not the most important ratio concerning the first shape PC.

212

213 For the species pair M. amaenus and Mesopolobus sp. n. the LDA ratio extractor identified stv.l/lgt.l

214 and hea.l/stv.l as the first two best discriminating ratios. These two combined ratios successfully

215 separated the two species (Figure 3a). The calculated ratios for the LDA-suggested characters (M.

216 amaenus vs Mesopolobus sp. n., range, mean, sd) are: stv.l/lgt.l (1.38-4.25, 2.38, 1.21) vs (0.80- 9

217 1.15, 0.92, 0.11); hea.l/stv.l (0.11-1.12, 0.93, 0.13) vs (1.11-1.53, 1.26, 0.11). This suggests that the

218 two species can be separated when judgement is based on a series of individuals. Further, the

219 calculated D.shape is much higher than D.size in all of the two best discriminative ratios, indicating

220 that species are mostly separated by differences in shape of characters (Table 3).

221

222 For the species pair M. fasciiventris and Mesopolobus sp. n. the LDA ratio extractor identified

223 pcl.l/mav.l and clv.l/hea.l as the first two best discriminating ratios. These two combined ratios

224 successfully separated the two species (Figure 3b). The calculated ratios for the LDA-suggested

225 characters (M. fasciiventris vs Mesopolobus sp. n., range, mean, sd) are: pcl.l3/mav.l (0.07-0.10,

226 0.08, 0.02) vs (0.11-0.20, 0.15, 0.05); clv.l/hea.l (0.36-0.66, 0.50, 0.14) vs (0.52-0.68, 0.63, 0.04).

227 This suggests that the two species can be separated when judgement is based on a series of

228 individuals. Further, the calculated D.shape is much higher than D.size in all of the two best

229 discriminative ratios, indicating that species are mostly separated by differences in shape of

230 characters (Table 3).

231

232 Because M. verditer and M. sericeus specimens were overlapping in the shape PCA with M.

233 amaenus we calculated the best ratios for discriminating M. amaenus from Mesopolobus sp. n. for

234 these two species as well. M. typographi overlapped in the shape PCA with M. fasciiventris, so we

235 also calculated the best ratios discriminating M. fasciiventris from Mesopolobus sp. n. for M.

236 typographi (Table 3).

237

238 Molecular species delimitation

239

240 Based on molecular analysis of the available samples, M. robiniae sp. n. is placed closest to

241 Mesopolobus verditer (Norton, 1868) (Figure 4). The three individuals represented only one

242 haplotype (653 bp) that was deposited in GenBank with the MF098549 accession number. The

243 alignment of the downloaded sequences was 468 bp long and consisted of 3 Pteromalus species

244 (used as outgroup) and 14 Mesopolobus species, including the one described in this paper.

245

246 The phylogenetic relationship between the species is unresolved based on the available COI

247 sequence data, but the tree shows a well-supported differentiation (PP=1) of the new species, with

248 M. verditer as the closest species (Figure 4). The differentiation is also supported by the p-distance

249 values with a minimum of 12.5% between M. robiniae sp. n. and M. verditer, and a maximum of

250 16.52% between M. robiniae sp. n. and M. tibialis (Table 4).

251

252 Taxonomy

253

254 Mesopolobus Westwood, 1833

255 Westwood, 1833, Philosophical Magazine (3) 2:443

256 Type species: Mesopolobus fasciiventris Westwood, by monotypy

257

258 Mesopolobus robiniae Lakatos & László, sp. n.

259 Figure 1. Female: 1a-b, e-f, i, k. Male: 1c-d, g-h, j, l.

260

261 Material examined:

262 Holotype, ♀, collected on 11.03.2015 near Săldăbagiu de Munte, Bihor County, 47.096354°N

263 21.984963°E by Lakatos, T. K, emerged on 01.04.2015, deposited in MZBBU id: MZBBU

264 HYM000011.

265

266 Paratypes, 1♂, collected on 11.03.2015 near Săldăbagiu de Munte, Bihor County, 47.096354°N

267 21.984963°E by Lakatos, T. K, emerged on 01.04.2015, deposited in MZBBU id: MZBBU

268 HYM000012; 1♀, collected on 17.03.2015 near Cluj-Napoca, Cluj County, 46.777109°N

269 23.674495°E by Lakatos, T. K, emerged on 10.04.2015, MZBBU id: MZBBU HYM000013; 1♀,

270 collected on 18.03.2014 near Cluj-Napoca, Cluj County, 46.777109°N 23.674495°E by Lakatos, T.

271 K, emerged on 05.2014, MZBBU id: MZBBU HYM000014; 2♀, collected on 17.03.2009 in Cluj-

272 Napoca, Cluj County, 46.768086°N 23.568935°E by Lakatos, T. K, emerged on 04.2009, MZBBU

273 id: MZBBU HYM000015 and HYM000016; 1♀, collected on 08.03.2014 near Săldăbagiu de

274 Munte, Bihor County, 47.100895°N E21.967509°E by Lakatos, T. K, emerged on 22.04.2014,

275 MZBBU id: MZBBU HYM000017; 1♀, collected on 11.03.2014 near Săldăbagiu de Munte, Bihor

276 County, 47.098182°N E21.975352°E by Lakatos, T. K, emerged on 23.04.2014, MZBBU id:

277 MZBBU HYM000018; 2♂, collected on 08.03.2014 near Săldăbagiu de Munte, Bihor County,

278 47.098182°N 21.975352°E by Lakatos, T. K, emerged on 22.04.2014, MZBBU id: MZBBU

279 HYM000019 and MZBBU HYM000020; 1♂, collected on 14.03.2009 near Săldăbagiu de Munte,

280 Bihor County, 47.079446°N 21.970817°E by Lakatos, T. K, emerged on 05.2009, MZBBU id:

281 MZBBU HYM000021; 1♂, collected on 11.03.2015 near Săldăbagiu de Munte, Bihor County,

282 47.098519°N 21.984808°E by Lakatos, T. K, emerged on 04.2015, MZBBU id: MZBBU

283 HYM000022; 1♂, collected on 02.03.2015 near Debrecen, Hajdú-Bihar County, Hungary,

284 47.554773°N 21.591610°E by Lakatos, T. K, emerged on 04.2015, MZBBU id: MZBBU

285 HYM000023; 1♂, collected on 22.03.2014 near Cluj-Napoca, Cluj County, 46.834976°N 12

286 23.651004°E by Lakatos, T. K, emerged on 05.2014, MZBBU id: MZBBU HYM000024; 1♂,

287 collected on 17.03.2009 in Cluj-Napoca, Cluj County, 46.768086°N 23.568935°E by Lakatos, T. K,

288 emerged on 05.2009, MZBBU id: MZBBU HYM000025.

289

290 The specimens used for the genetic analysis were collected on 13.03.2014 near Săldăbagiu de

291 Munte, Bihor County, 47.0968°N 21.98525°E and emerged on 17.04.2014.

292

293 Description of Mesopolobus robiniae Lakatos & László, sp. n.

294

295 FEMALE. Length 2.05 to 3.00 mm (N=15, mean=2.6, sd=0.29 mm).

296

297 Coloration. Body green, sometimes with golden reflections; gaster bronze-black distally, some of

298 the tergites occasionally with blue or violet flecks. Coloration of antennae: scape, pedicellus and

299 anelli testaceous, sometimes last anellus infuscate, all funicular segments and clava always

300 infuscate, occasionally brown. Coxae concolorous with the thorax, femora and tibiae testaceous, the

301 tips of the fifth tarsi fuscous to black. Tegulae hyaline, usually slightly yellow posteriorly. Wings

302 hyaline; venation pale yellow.

303

304 Head 1.1 (range 1.02-1.18) times as broad as mesoscutum; in dorsal view 2.25 (2.07-2.52) times as

305 broad as long, with temples rounded off and between one third and one fourth as long as eyes; POL

306 2.11 (1.75-2.80) times OOL. Head in front view suboval with the genae moderately buccate. Eyes

307 separated about 1.59 (1.18-1.74) times their length. Malar space more than half (0.68 (0.55-0.76))

308 the length of an eye. Breadth of oral fossa 1.93 (1.69-2.36) times the malar space. Clypeus strigose,

309 its anterior margin moderately emarginate. Head uniformly and moderately reticulate. Antennae 13

310 inserted low on head, lower edge of toruli at or hardly above level of ventral edge of eyes; distance

311 between clypeal margin and toruli 0.69 (0.54-0.8) times the distance between median ocellus and

312 toruli. Scape length 1.23 (1.09-1.4) times eye length, scape almost reaching lower edge of median

313 ocellus; combined length of pedicellus and flagellum 0.87 (0.76-0.96) times breadth of head;

314 pedicellus (profile) 2.06 (0.75-2.5) times long as broad, about as long as anelli plus first funicular

315 segment; flagellum rather weakly clavate, proximally as stout as or slightly stouter than the

316 pedicellus; first and second anelli short, twice or rather more than twice as broad as long, third

317 anellus longer and about 1.5 times as broad as long; funicular segments subquadrate, the proximal

318 ones sometimes slightly longer than broad, the distal ones occasionally very slightly transverse;

319 clava 1.9 (1.5-2.29) times long as broad, 0.83 (0.66-1.15) as long as the three preceding funicular

320 segments together; sensilla in one row on each segment, sparse on the funicle, more numerous on

321 the clava.

322

323 Mesosoma 1.52 (1.38-1.74) times as long as broad. Pronotal collar moderately long medially, 0.21

324 (0.16-0.26) (one sixth to one fifth) as long as mesoscutum, and much longer at the sides, strongly

325 and coarsely reticulate, clearly margined. Mesoscutum 1.58 (1.28-1.82) times as broad as long,

326 rather coarsely reticulate discally, more finely laterally, without piliferous punctures. Scutellum 0.9

327 (0.82-0.94) as broad as long, moderately convex, finely reticulate, the frenum rather more coarsely

328 reticulate. Axillae finely reticulate. Dorsellum a narrow, alutaceous transverse crest which is

329 separated from the scutellum by a simple suture. Propodeum medially slightly less than half (0.41

330 (0.36-0.48)) as long as the scutellum; median area 2.39 (2-3) times as broad as long, well-defined

331 laterally, the plicae distinct throughout and sharp over at least their distal half; median carina

332 distinct, straight; panels of median area finely, slightly irregularly reticulate; nucha transversely

333 aciculate, separated from the median area by an impressed line; posterior foveae, at sides of nucha, 14

334 moderately deep; spiracles oval, longer than broad, separated by nearly half their length from the

335 metanotum. Postspiracular sclerite broad, shiny, weakly and irregularly sculptured. Mesepisternum

336 moderately finely reticulate, its upper triangular area smooth; mesepimeron rather more coarsely

337 reticulate than the mesepisternum, metapleuron smooth. Legs rather short; femora rather stout; mid

338 tibiae fairly slender, 7.44 (4.88-9) as long as their maximum breadth. Fore wing rather broad; costal

339 cell fairly broad, its upper surface bare, lower surface with a complete row of hairs and some

340 additional hairs scattered over the distal third to half; basal cell bare, open below; basal vein bare or

341 with one to three hairs; speculum open below, on upper surface of wing extending below the

342 proximal end of the marginal vein; surface beyond the speculum thickly pilose; marginal vein 2.19

343 (2-2.47) times as long as the stigmal vein; postmarginal vein shorter than the marginal, 0.73 (0.63-

344 0.81) times as long as the marginal.

345

346 Gaster ovate, 1.24 (1.16-1.33) times longer than mesosoma, 0.8 (0.66-0.96) times broader than

347 mesosoma, 2.37 (1.91-2.96) times as long as broad; basal tergite occupying from slightly more than

348 one quarter, to nearly one third, the total length; last tergite somewhat shorter than its basal breadth,

349 its length 1.07 (0.72-1.79) times its breadth; ovipositor sheaths projecting at most very slightly;

350 hypopygium slightly reaching the middle of the gaster, ratio of hypopygium length to gaster length

351 is 0.44 (0.35-0.54).

352

353 MALE. Length 1.8 to 2.25 mm (N=15, mean=2.02, sd=0.16 mm).

354

355 Coloration: head and mesosoma bright green; gaster greenish dorsally with T2 and posterior half of

356 TI yellow, T3 purplish; antennae bright testaceous; legs except coxae yellow, last tarsal segments

357 grey-brown. 15

358

359 Head: antenna with 3 anelli and 5 funicular segments, length of pedicel plus flagellum 0.97 (0.85-

360 1.04) times breadth of head; scape 5.33 (4.6-6.25) times as long as broad, without a boss on its

361 anterior surface; flagellum proximally not broader than pedicel, F1-F4 longer than broad, F5

362 subquadrate. Mouthparts unmodified; no patch of modified sculpture behind malar sulcus.

363

364 Mesosoma: Pronotal collar long as in females, about 0.21 (0.18-0.27) mesoscutal length. Middle

365 tibiae unmodified, middle tibia 7.42 (6.29-8.8) times its breadth, tibial spur 1.47 (1.17-1.8) times

366 breadth of first tarsus.

367

368 Gaster oblong, ovate, 0.91 (0.83-1.02) times as long as mesosoma, 2.24 (1.63-2.63) times as long as

369 broad with a yellow ventral plica; T1 with triangular depression at base.

370

371 Etymology

372

373 The new Mesopolobus species is named after the host plant of its seed predator host, the black locust

374 (Robinia pseudoacacia).

375

376 Diagnosis

377 Morphological comparison

378

379 M. robiniae sp. n. females were not identifiable based on Graham’s keys (Graham, 1969), but

380 several morphologically and morphometrically related species were found for which the differing

381 characters will be enumerated in the order that the species appear in Graham’s key. The species M. 16

382 robiniae sp. n. has a shorter gaster compared to head plus thorax than M. maculicornis. The species

383 M. jucundus has a curved stigmal vein compared to M. robiniae sp. n. Mesopolobus robiniae sp. n.

384 differs from M. fasciiventris by its males having 3 anelli and 5 funicular segments while in the latter

385 there are 2 anelli and 6 segments. The head of M. apicalis in dorsal view has temples nearly three

386 quarters as long as the eyes, while M. robiniae sp. n. has its head in dorsal view temples appearing

387 one quarter to one third as long as the eyes. The gaster of M. amaenus is less than twice as long as

388 its breadth and almost as long as the thorax, while in the case of M. robiniae sp. n gaster is not less

389 than twice as long as broad, but it is as long as thorax. The species M. longicollis has the pronotal

390 collar 1/7 to 1/6 as long as the mesoscutum and its gaster is less than twice as long as broad

391 compared to M. robiniae sp. n. The species M. diffinis and M. meditteraneus differ from M. robiniae

392 sp. n. because the latter has longer marginal vein as 1.4 to 1.6 as length of the stigmal vein.

393

394 The species M. verditer is not present in the keys of Graham (1969) because it has a North-

395 American distribution. It differs from M. robiniae sp. n. in the following: antennal funicle segments

396 shorter than their length, while in M. robiniae sp. n. they are at least as long as their breadth. The

397 ratio of the stigma vein to the last gastral tergite length is 1.91-2.50 in M. verditer, while in M.

398 robiniae sp. n. is between 0.08-1.15. Mespolobus sericeus differs from M. robiniae sp. n. first by

399 having 2 anelli and 6 funicular segments, but also in having the ratio of the stigmal vein to the last

400 gastral tergite length 1.41, while in the other species this ratio is smaller (0.8-1.15). From M.

401 typographi the species M. robiniae sp. n. differs in the ratio of the pronotal collar length to the

402 marginal vein length which in the first species is 0.1 (N=1) and in the second is between 0.11-0.02

403 (N=15). Moreover, in M. typographi the median area of propodeum is 1.75-2 times as broad as long

404 (Graham 1969) while in M. robiniae sp. n. is 0.82-0.94 times as broad as long (N=15).

405 17

406 Based on von Rosen’s key (von Rosen, 1958), the morphological identification of specimens led us

407 to M. mediterraneus (Mayr, 1903) as the closest species, from which M. robiniae sp. n. females

408 differed in having a longer pronotal collar, much longer marginal than stigmal vein and a shorter

409 gaster than the combined length of head and mesosoma.

410

411 In Gahan (1932) page 739 says that Mesopolobus (syn. Amblymerus) verditer (Norton, 1868) “…

412 conforms very closely to the characters of the genus Amblymerus Walker as represented by

413 Amblymerus amoenus Walker…” (syn. M. amaenus), when transferring the species to genus

414 Amblymerus Walker, 1834 from the genus Nasonia Ashmead, 1904. The hosts of M. verditer are

415 usually sawflies (Hymenoptera: Diprionidae) on pines (Pinus sp.) (Noyes, 2020). M. verditer is

416 distributed in the Nearctic and Germany (W. R. Thompson, 1958). Moreover, M. verditer differs

417 from M. robiniae sp. n. in having a reticulated middle area of propodeum and oblique wrinkles, as

418 does also from M. amaenus and M. longicollis (von Rosen, 1958).

419

420 We propose the following update to the key of Mesopolobus species of Graham (1969) for females:

421

422 16(14) - Either gaster at least slightly longer than head plus thorax, and usually more than twice as

423 long as broad, or gaster not longer than head plus thorax, and at most twice as long as broad.

424 ……………………………………….…………………………………………….…….. 16A

425 – Gaster not longer than head plus thorax, their ratio is 0.94 (0.88-0.98), gaster usually more

426 than twice, 2.37 (1.91-2.96) as long as broad …………………………….. M. robiniae sp. n.

427 16A(16) - Gaster at least slightly longer than head plus thorax, usually more than twice as long as

428 broad……………………………………………………………………………………….. 17

429 – Gaster not longer than head plus thorax, at most twice as long as broad …………………. 27 18

430

431 Distribution

432

433 The type locality for M. robiniae sp. n. is Săldăbagiu de Munte, Bihor County, Romania

434 (N47.096354 E21.984963). The other localities are situated in the neighbouring counties: Cluj

435 County, Romania and Hajdú-Bihar County, Hungary. The species may appear in the Carpathian

436 Basin where its host plant is present, but we expect that it may also be found outside of the

437 Carpathian Basin, in Eastern Europe and maybe throughout Europe.

438

439 Biology

440

441 Based on our rearing, M. robiniae sp. n. seems to be an early flying parasitoid species. Individuals

442 of the species emerged during spring consequently in all study years. Our black locust seedpod

443 samples were collected mostly in March, and the peak of M. robiniae emergence was in April, with

444 a decrease in May. After May we rarely encountered any individuals of this parasitoid species.

445

446 The host of M. robiniae sp. n. may be Bruchophagus robiniae but there is no information regarding

447 the host plant of B. robiniae before the introduction of black locust. Another possibility is that M.

448 robiniae sp. n. initially had another host, but has switched from it to B. robiniae. Either possibility is

449 plausible; before 1970 (Zerova, 1970) the species B. robiniae was not known, and M. robiniae sp. n.

450 was not described until now. The parasitoid community of black locust is understudied, and the

451 available literature makes no mention of parasitoids in this community (Farkas & Terpó-Pomogyi,

452 1974; Perju, 1998), with the exception of our ecological study concerning the seed-predator

453 community of black locust in Eastern Europe (Lakatos et al., 2016). 19

454

455 Discussion

456

457 The multivariate ratio analysis (MRA) and the mtDNA sequence analysis resulted in the successful

458 separation of the Mesopolobus species emerging from black locust seedpods from the other

459 congeneric relatives. The morphometry-based shape PCA helped us identify which species fall

460 closer to the specimens emerged from black locust crops. This delimitation was important since the

461 available specific keys (Graham, 1969) did not lead us to a closest relative based on the combination

462 of morphology and morphometry.

463

464 In a PCA ratio spectrum, only ratios calculated with variables lying at the opposite ends of the

465 spectrum are relevant for a particular shape PC and the most allometric ratios are also found at the

466 opposite ends of the allometry ratio spectrum (Baur et al., 2014). The PCA ratio spectrum and the

467 allometry ratio spectrum plots revealed a large (M. amaenus and M. robiniae sp. n.) and moderate

468 (M. fasciiventris and M. robiniae sp. n.) amount of allometric variation in the identified

469 discriminating morphometric character pairs (Figure 2). However, this is not of concern in our case,

470 because on one hand the species we found to be closely related based on morphometry were clearly

471 separated based on the molecular results, and the combinations of the usually used ratios do not

472 overlap with species in the keys of Graham, since there is no possibility to progress beyond key

473 couplet 16. On the other hand, the ratios found with the LDA ratio extractor tool have small D.size

474 values compared to D.shape values (Table 3) which means that separation was mainly due to shape

475 rather than size. The LDA ratio extractor tool found that the species pairs could be separated without

476 overlapping based on the first ratio pairs. These ratios in combination with morphologic characters

477 gave a confident separation of the closely related species. 20

478

479 The origin of M. robiniae sp. n. species is yet unknown, since it has to be a host shifting species.

480 Black locust was introduced to Europe 300 years ago, and in its native area it has no Bruchophagus

481 seed predator, nor the associated parasitoids (Stone, 2009). So, the new Mesopolobus species may

482 not be monophagous on B. robiniae, which is similarly a host-shifting seed predator. Nonetheless, it

483 is befitting of the name robiniae, since parasitoids are also affected by the host plant of their

484 herbivorous host. As part of their host finding strategy, parasitoids may search for a specific plant or

485 plant part (as seedpods) housing any potential herbivorous host species (Cronin & Abrahamson,

486 2001).

487

488 Acknowledgements

489

490 We thank to Zoltán Vas, Curator of Hymenoptera collection, Hungarian Natural History Museum

491 for loaning several specimens of various Mesopolobus species and for his valuable help during

492 identification and manuscript preparation. We are thankful to Natalie Dale-Skey, curator of the

493 Hymenoptera section, Natural History Museum for loaning several Mesopolobus specimens and to

494 James Hogan Collections Manager of Hope Entomological Collections, Oxford University Museum

495 of Natural History for providing photography of M. longicollis. We are also thankful to Lajos Király

496 for his help in the molecular analysis. The authors are grateful to Chris Looney (Washington State

497 Department of Agriculture, Olympia, United States) for his review, comments and suggestions of

498 the manuscript. Molecular analysis was done at the Interdisciplinary Research Institute on Bio–

499 Nano–Sciences of BBU, Cluj, Romania. During preparation of the manuscript AL Dénes received

500 financial support from the Collegium Talentum scholarships, Hungary.

501 21

502 Disclosure

503 The authors declare that they have no conflict of interests.

504

505 Author contributions

506

507 LZ and LKT designed the study. LKT collected data, made morphometric measurements,

508 participated in paper writing. DAL made molecular analyses, participated in paper writing. LZ

509 analyzed, interpreted data and drafted the manuscript. All authors gave final approval for

510 publication.

511

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620

621 Table 1. Collection dates, location and number of M. robiniae sp. n. individuals. BH: Bihor County,

622 Romania, CJ: Cluj County, Romania, HB: Hajdú-Bihar County, Hungary.

Collection date Location Northing Easting N N male N female

2009 BH 47.098202 21.975355 9 7 2

2009 CJ 46.827366 23.629258 44 16 28

2013 BH 47.098202 21.975355 4 0 4

2013 CJ 46.801433 23.611995 26 12 14

2014 BH 47.098202 21.975355 69 35 34

2014 CJ 46.801433 23.611995 104 62 42

2014 HB 47.554773 21.591610 58 48 10

2015 BH 47.098202 21.975355 16 8 8

2015 CJ 46.801433 23.611995 23 13 10

2015 HB 47.554773 21.591610 30 14 16

23 Table 2. The selected morphometric characters of female Mesopolobus specimens, abbreviations used, and their description. bioRxiv preprint

Abbreviation Character name Definition Magnification was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade clv.b Clava breadth Greatest breadth of antennal clava, lateral view 80× clv.l Clava length Length of antennal clava, lateral view 80× hea.b Head breadth Greatest breadth of head, dorsal view 60× doi: hea.l Head length Head length in dorsal view (Graham, 1969), distance between anterior and posterior margin of the 60× https://doi.org/10.1101/2020.05.01.072140 head, measured laterally ltg.b Seventh gastral Greatest breadth of the seventh gastral tergite, greatest distance between the outermost lateral edges 80× tergite breadth of the seventh gastral tergite ltg.l Seventh gastral Greatest length of the seventh gastral tergite, greatest distance between the anterior and posterior 80× tergite length edges of the seventh gastral tergite mv.l Marginal vein Length of marginal vein, distance between the point at which the submarginal vein touches the 80× available undera leading edge of the wing and the point at which stigmal vein and postmarginal vein unite (Graham, 1969) msc.b Mesoscutum Greatest breadth of mesoscutum just in front of level of tegula, dorsal view 80×

breadth CC-BY-NC-ND 4.0Internationallicense msc.l Mesoscutum Length of mesoscutum along median line from posterior edge of pronotum to posterior edge of 80× ; length mesoscutum, dorsal view this versionpostedMay2,2020. mss.l Mesosoma length Length of mesosoma along median line from anterior edge of pronotum collar to posterior edge of 60× nucha, dorsal view ool.l OOL Shortest distance between posterior ocellus and eye margin, dorsal view (Graham, 1969) 80× pcl.l Pronotal collar Length of pronotal collar along the median line from the edge between neck and pronotal collar to 80× length anterior edge of mesoscutum, dorsal view ped.b Pedicellus breadth Greatest breadth of pedicel, dorsal view 80× ped.l Pedicellus length Greatest length of pedicel, dorsal view 80× pol.l POL Shortest distance between posterior ocelli, dorsal view (Graham, 1969) 80× The copyrightholderforthispreprint(which ppd.l Propodeum length Length of propodeum measured along median line from anterior edge to posterior edge of nucha, 80× . dorsal view sct.b Scutellum breadth Greatest breadth of the scutellum, greatest distance between the outermost lateral edges of the 80× scutellum sct.l Scutellum length Length of scutellum along median line from posterior edge of mesoscutum to posterior edge of 80× scutellum, dorsal view stv.l Stigmal vein Length of stigmal vein, distance between the point at which stigmal vein and postmarginal vein 80× unite apically, and the distal end of the stigma (Graham, 1969) 24

25 Table 3. First and second-best ratios found by the LDA ratio extractor for separating various groups and specimens of Mesopolobus females. bioRxiv preprint

Best ratios Range group 1 Range group 2 D.shape D.size was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade

Group / species comparison doi:

M. amaenus vs. Mesopolobus sp. n. stv.l/lgt.l 1.38-4.25 0.80-1.15 0.73 0.03 https://doi.org/10.1101/2020.05.01.072140

hea.l/stv.l 0.11-1.12 1.11-1.53 0.72 0.04

M. verditer stv.l/lgt.l 1.91-2.50 available undera hea.l/stv.l 1.14

M. sericeus stv.l/lgt.l 1.41

hea.l/stv.l 1.1 5 CC-BY-NC-ND 4.0Internationallicense ; this versionpostedMay2,2020. M. fasciiventris vs. Mesopolobus sp. n. pcl.l/mav.l 0.07-0.10 0.11-0.20 0.65 0.01

clv.l/hea.l 0.36-0.66 0.52-0.68 0.63 0.01

M. typographi pcl.l/mav.l 0.1

clv.l/hea.l 0.55 The copyrightholderforthispreprint(which 26 .

28 Table 4. P-distance values for sequences of M. robiniae sp. n. and all available Mesopolobus species. bioRxiv preprint

M. verditer was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade

M. bruchophagi 12.66 doi:

M. amaenus 12.71 12.46 https://doi.org/10.1101/2020.05.01.072140

M. tortricis 14.22 13.08 14.98

M. dubius 15.41 13.54 14.89 17.66 available undera M. lichtensteini 13.47 13.68 10.06 15.98 14.02

M. sericeus 14.75 13.87 14.46 15.6 15.81 14.45

M. fuscipes 13.22 12.39 13.87 15.75 13.89 13.16 11.82 CC-BY-NC-ND 4.0Internationallicense ; this versionpostedMay2,2020. M. fasciiventris 16.98 14.73 14.96 15.58 15.15 15.1 14.65 16.65

M. tibialis 15.46 11.2 15.46 16.82 14.39 15.74 14.81 14.25 16.08

M. xanthocerus 15.51 12.4 13.44 17.28 12.18 14.74 12.89 11.54 15.08 12.11

M. morys 12.68 10.48 12.76 14.18 13.66 13.15 13.9 10.89 14.69 13.9 12.03 The copyrightholderforthispreprint(which M. robiniae sp. n. 12.5 13.54 14.39 14.68 14.21 15.02 14.39 15.81 15.94 16.52 15.17 15.85 .

629 Figure legends

630

631 Figure 1. Scatterplot of first against second shape PC based on 19 morphometric variables of

632 females of a) all 14 Mesopolobus species b) M. amaenus and Mesopolobus sp. n emerged from

633 black locust seedpods c) M. fasciiventris and Mesopolobus sp. n. emerged from black locust

634 seedpods. The variance explained by each shape PC is given in parentheses.

635

636 Figure 2. Ratio spectra for the two species pairs: a) and b) show M. amaenus and Mesopolobus sp.

637 n, while c) and d) show M. fasciiventris and Mesopolobus sp. n. The left figures a) and c) show the

638 PCA ratio spectrum, while the ones on right side b) and d) show the allometry ratio spectrum;

639 horizontal bars in the ratio spectra represent 68% bootstrap confidence intervals based on 1000

640 replicates.

641

642 Figure 3. Scatterplots of the two most discriminating ratios for females a) of M. amaenus and

643 Mesopolobus sp. n and b) M. fasciiventris and Mesopolobus sp. n. Both plots show first versus

644 second ratio from LDA ratio extract analysis.

645

646 Figure 4. Bayesian inference (BI) tree of the Mesopolobus species that have available mitochondrial

647 COI sequences. Numbers on the branches represent posterior probabilities (PP).

648

649 Figure 5. a) female head, frontal view b) female antenna c) male head, frontal view d) male antenna

650 e) female mesosoma f) female fore wing g) male mesosoma h) male fore wing i) female gaster,

651 dorsal view j) male gaster, dorsal view k) female habitus l) male habitus of Mesopolobus robiniae

652 sp. n. 32 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.01.072140; this version posted May 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.